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Thromb Haemost 2015; 114(02): 325-336
DOI: 10.1160/TH14-11-0981
Cellular Haemostasis and Platelets
Schattauer GmbH

Functional role of a polymorphism in the Pannexin1 gene in collageninduced platelet aggregation

Filippo Molica
1   Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland
2   Department of Medical Specializations – Cardiology, University of Geneva, Geneva, Switzerland
,
Sandrine Morel   $ and *
1   Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland
,
Merlijn J. Meens   $ and *
1   Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland
2   Department of Medical Specializations – Cardiology, University of Geneva, Geneva, Switzerland
,
Jean-François Denis
1   Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland
2   Department of Medical Specializations – Cardiology, University of Geneva, Geneva, Switzerland
,
Paul F. Bradfield
1   Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland
,
Silvia Penuela
3   Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario, Canada
,
Anne Zufferey
4   Division of Angiology and Haemostasis, University Hospitals of Geneva and Geneva Platelet Group, University of Geneva, Geneva, Switzerland
,
Hannah Monyer
5   Department of Clinical Neurobiology, University of Heidelberg, Heidelberg, Germany
,
Beat A. Imhof
1   Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland
,
Marc Chanson
6   Department of Pediatrics, Geneva University Hospitals and University of Geneva, Geneva, Switzerland
,
Dale W. Laird
3   Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario, Canada
,
Pierre Fontana   $ and *
4   Division of Angiology and Haemostasis, University Hospitals of Geneva and Geneva Platelet Group, University of Geneva, Geneva, Switzerland
,
Brenda R. Kwak   $ and *
1   Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland
2   Department of Medical Specializations – Cardiology, University of Geneva, Geneva, Switzerland
› Author Affiliations Financial support:This work was supported by grants from the Swiss National Science Foundation (310030_143343 to B. R. Kwak and 320030_144150 to P. Fontana).
Further Information

Publication History

Received: 25 November 2014

Accepted after major revision: 16 March 2015

Publication Date:
21 November 2017 (online)

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Summary

Pannexin1 (Panx1) forms ATP channels that play a critical role in the immune response by reinforcing purinergic signal amplification in the immune synapse. Platelets express Panx1 and given the importance of ATP release in platelets, we investigated Panx1 function in platelet aggregation and the potential impact of genetic polymorphisms on Panx1 channels. We show here that Panx1 forms ATP release channels in human platelets and that inhibiting Panx1 channel function with probenecid, mefloquine or specific 10Panx1 peptides reduces collagen-induced platelet aggregation but not the response induced by arachidonic acid or ADP. These results were confirmed using Panx1-/- platelets. Natural variations have been described in the human Panx1 gene, which are predicted to induce non-conservative amino acid substitutions in its coding sequence. Healthy subjects homozygous for Panx1–400C, display enhanced platelet reactivity in response to collagen compared with those bearing the Panx1–400A allele. Conversely, the frequency of Panx1–400C homozygotes was increased among cardiovascular patients with hyper-reactive platelets compared with patients with hypo-reactive platelets. Exogenous expression of polymorphic Panx1 channels in a Panx-deficient cell line revealed increased basal and stimulated ATP release from cells transfected with Panx1–400C channels compared with Panx1–400A expressing transfectants. In conclusion, we demonstrate a specific role for Panx1 channels in the signalling pathway leading to collagen-induced platelet aggregation. Our study further identifies for the first time an association between a Panx1–400A>C genetic polymorphism and collagen-induced platelet reactivity. The Panx1–400C variant encodes for a gain-of-function channel that may adversely affect atherothrombosis by specifically enhancing collagen-induced ATP release and platelet aggregation.

Keywords

Pannexin - platelet aggregation - ATP - gene polymorphism

$ and * Authors have contributed equally to this study.


 

  • References

  • 1 Go AS, Mozaffarian D, Roger VL. et al. Heart disease and stroke statistics--2014 update: a report from the American Heart Association. Circulation 2014; 129: e28-e292.
    CrossrefPubMedGoogle Scholar
  • 2 Mackman N. Triggers, targets and treatments for thrombosis. Nature 2008; 451: 914-918.
    CrossrefPubMedGoogle Scholar
  • 3 Brass LF, Zhu L, Stalker TJ. Minding the gaps to promote thrombus growth and stability. J Clin Invest 2005; 115: 3385-3392.
    CrossrefPubMedGoogle Scholar
  • 4 Jackson SP. Arterial thrombosis--insidious, unpredictable and deadly. Nature Med 2011; 17: 1423-1436.
    CrossrefPubMedGoogle Scholar
  • 5 Swieringa F, Kuijpers MJ, Heemskerk JW. et al. Targeting platelet receptor function in thrombus formation: The risk of bleeding. Blood Rev 2014; 28: 9-21.
    CrossrefPubMedGoogle Scholar
  • 6 Brass LF, Stalker TJ. Minding the gaps--and the junctions, too. Circulation 2012; 125: 2414-2416.
    CrossrefPubMedGoogle Scholar
  • 7 Penuela S, Gehi R, Laird DW. The biochemistry and function of pannexin channels. Biochim Biophys Acta 2013; 1828: 15-22.
    CrossrefPubMedGoogle Scholar
  • 8 Baroja-Mazo A, Barbera-Cremades M, Pelegrin P. The participation of plasma membrane hemichannels to purinergic signalling. Biochim Biophys Acta 2013; 1828: 79-93.
    CrossrefPubMedGoogle Scholar
  • 9 Sosinsky GE, Boassa D, Dermietzel R. et al. Pannexin channels are not gap junction hemichannels. Channels 2011; 05: 193-197.
    CrossrefPubMedGoogle Scholar
  • 10 Penuela S, Simek J, Thompson RJ. Regulation of pannexin channels by post-translational modifications. FEBS Lett. 2014 Epub ahead of print.
    PubMedGoogle Scholar
  • 11 Penuela S, Harland L, Simek J. et al. Pannexin channels and their links to human disease. Biochem J 2014; 461: 371-381.
    CrossrefPubMedGoogle Scholar
  • 12 Chekeni FB, Elliott MR, Sandilos JK. et al. Pannexin 1 channels mediate ’findme’ signal release and membrane permeability during apoptosis. Nature 2010; 467: 863-867.
    CrossrefPubMedGoogle Scholar
  • 13 Chen Y, Yao Y, Sumi Y. et al. Purinergic signalling: a fundamental mechanism in neutrophil activation. Science Signal 2010; 03: ra45.
    PubMedGoogle Scholar
  • 14 Bao Y, Chen Y, Ledderose C. et al. Pannexin 1 channels link chaemoattractant receptor signalling to local excitation and global inhibition responses at the front and back of polarized neutrophils. J Biol Chem 2013; 288: 22650-22657.
    CrossrefPubMedGoogle Scholar
  • 15 Woehrle T, Yip L, Elkhal A. et al. Pannexin-1 hemichannel-mediated ATP release together with P2X1 and P2X4 receptors regulate T-cell activation at the immune synapse. Blood 2010; 116: 3475-3484.
    CrossrefPubMedGoogle Scholar
  • 16 Schenk U, Westendorf AM, Radaelli E. et al. Purinergic control of T cell activation by ATP released through pannexin-1 hemichannels. Science Signal 2008; 01: ra6.
    PubMedGoogle Scholar
  • 17 Yip L, Woehrle T, Corriden R. et al. Autocrine regulation of T-cell activation by ATP release and P2X7 receptors. FASEB J 2009; 23: 1685-1693.
    CrossrefPubMedGoogle Scholar
  • 18 Taylor KA, Wright JR, Vial C. et al. Amplification of human platelet activation by surface pannexin-1 channels. J Thromb Haemost. 2014 Epub ahead of print.
    PubMedGoogle Scholar
  • 19 Angelillo-Scherrer A, Fontana P, Burnier L. et al. Connexin 37 limits thrombus propensity by downregulating platelet reactivity. Circulation 2011; 124: 930-939.
    CrossrefPubMedGoogle Scholar
  • 20 Zufferey A, Schvartz D, Nolli S. et al. Characterisation of the platelet granule proteome: evidence of the presence of MHC1 in alpha-granules. J Proteomics 2014; 101: 130-140.
    CrossrefPubMedGoogle Scholar
  • 21 Mahaut-Smith MP, Jones S, Evans RJ. The P2X1 receptor and platelet function. Purin Signal 2011; 07: 341-356.
    PubMedGoogle Scholar
  • 22 Bargiotas P, Krenz A, Hormuzdi SG. et al. Pannexins in ischaemia-induced neurodegeneration. Proc Natl Acad Sci USA 2011; 108: 20772-20777.
    CrossrefPubMedGoogle Scholar
  • 23 Penuela S, Bhalla R, Gong XQ. et al. Pannexin 1 and pannexin 3 are glycoproteins that exhibit many distinct characteristics from the connexin family of gap junction proteins. J Cell Sci 2007; 120: 3772-3783.
    CrossrefPubMedGoogle Scholar
  • 24 Fontana P, Nolli S, Reber G. et al. Biological effects of aspirin and clopidogrel in a randomized cross-over study in 96 healthy volunteers. J Thromb Haemost 2006; 04: 813-819.
    CrossrefPubMedGoogle Scholar
  • 25 Zufferey A, Reny JL, Combescure C. et al. Platelet reactivity is a stable and global phenomenon in aspirin-treated cardiovascular patients. Thromb Haemost 2011; 106: 466-474.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 26 Ransford GA, Fregien N, Qiu F. et al. Pannexin 1 contributes to ATP release in airway epithelia. Am J Resp Cell Mol Biol 2009; 41: 525-534.
    CrossrefPubMedGoogle Scholar
  • 27 Seminario-Vidal L, Okada SF, Sesma JI. et al. Rho signalling regulates pannexin 1-mediated ATP release from airway epithelia. J Biol Chem 2011; 286: 26277-26286.
    CrossrefPubMedGoogle Scholar
  • 28 Hechler B, Magnenat S, Zighetti ML. et al. Inhibition of platelet functions and thrombosis through selective or nonselective inhibition of the platelet P2 receptors with increasing doses of NF449 [4,4’,4’’,4’’’-(carbonylbis(imino-5,1,3-benzenetriylbis-(carbonylimino)))tetrakis -benzene-1,3-disulfonic acid octasodium salt]. J Pharmacol Exp Therap 2005; 314: 232-243.
    CrossrefPubMedGoogle Scholar
  • 29 Weilinger NL, Tang PL, Thompson RJ. Anoxia-induced NMDA receptor activation opens pannexin channels via Src family kinases. J Neurosci 2012; 32: 12579-12588.
    CrossrefPubMedGoogle Scholar
  • 30 Mahaut-Smith MP. The unique contribution of ion channels to platelet and megakaryocyte function. J Thromb Haemost 2012; 10: 1722-1732.
    CrossrefPubMedGoogle Scholar
  • 31 Vaiyapuri S, Jones CI, Sasikumar P. et al. Gap junctions and connexin hemichannels underpin haemostasis and thrombosis. Circulation 2012; 125: 2479-2491.
    CrossrefPubMedGoogle Scholar
  • 32 Derouette JP, Desplantez T, Wong CW. et al. Functional differences between human Cx37 polymorphic hemichannels. J Mol Cell Cardiol 2009; 46: 499-507.
    CrossrefPubMedGoogle Scholar
  • 33 Hechler B, Lenain N, Marchese P. et al. A role of the fast ATP-gated P2X1 cation channel in thrombosis of small arteries in vivo. J Exp Med 2003; 198: 661-667.
    CrossrefPubMedGoogle Scholar
  • 34 Oury C, Daenens K, Hu H. et al. ERK2 activation in arteriolar and venular murine thrombosis: platelet receptor GPIb vs. P2X. J Thromb Haemost 2006; 04: 443-452.
    CrossrefPubMedGoogle Scholar
  • 35 Gijsen F, van der Giessen A, van der Steen A. et al. Shear stress and advanced atherosclerosis in human coronary arteries. J Biomechan 2013; 46: 240-247.
    CrossrefPubMedGoogle Scholar
  • 36 Kalmatsky BD, Batir Y, Bargiello TA. et al. Structural studies of N-terminal mutants of connexin 32 using (1)H NMR spectroscopy. Arch Biochem Biophys 2012; 526: 1-8.
    CrossrefPubMedGoogle Scholar
  • 37 Zahid M, Mangin P, Loyau S. et al. The future of glycoprotein VI as an anti-thrombotic target. J Thromb Haemost 2012; 10: 2418-2427.
    CrossrefPubMedGoogle Scholar